Benzamides can stimulate as well as inhibit the activity of nuclear ADP-ribosyltransferase

Epithelial cells from the lens equator differentiate into elongated fiber cells. In the final steps of differentiation, the chromatin appears quite condensed and chromatin breakdown into nucleosomes occurs. DNA breaks due to an endodeoxyribonuclease activity corresponding to at least two polypeptides of 30 and 40 kDa have been identified. To identify the nature and the developmental appearance of initial breaks, nick translation reaction was followed both biochemically and in situ in fiber and epithelial cells from chick embryonic lenses. There is no accumulation of single-strand breaks (SSB) with 3'OH ends in lens fiber cells during embryonic development. Such damage can be increased in these cells by treatment with DNAase I indicating the absence of an inhibitor of the nick translation reaction in fiber cells. However, there are indications of the presence of DNA breaks with blocked termini when the phosphatase activity of nuclease P1 is used. The presence of breaks is also indicated by the large amounts of (ADP-ribose)n found in lens fibers particularly at 11 days of embryonic development (E11) as ADP-ribosyl transferase binds to and is activated by DNA strand breaks. Incubation of lens cells in vitro, which causes nucleosomal fragmentation only in fiber cells, produces SSB with 3'OH ends in both epithelia and fibers. Incubation for short periods, observed in experiments in situ, induces SSB first in the central fiber nuclei, which are late in differentiation. This may indicate that these SSB play a physiological role. Long incubations produce larger numbers of SSB in epithelia than fibers. The SSB in the fibers may have been converted into double-strand breaks (D SB), seen as nucleosomal fragments, and therefore no longer act as substrates for nick translation. The nuclease activity responsible for SSB production is independent of divalent cations and could be implicated in lens terminal differentiation.

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“…The methodology was that of Jacobson et al (1984) as described in Jones et al (1988). Each sample was first precipitated with cold 20% TCA.…”

Section: P1mentioning

confidence: 99%

“…Assay of (ADP-ribose), Epithelia or fiber masses (900-1,000) from chick lenses at E l l and E l 8 day were used to determine the amount of (ADP-ribose),. The methodology was that of Jacobson et al (1984) as described in Jones et al (1988). Each sample was first precipitated with cold 20% TCA.…”

Section: Treatment With Nuclease P1mentioning

confidence: 99%

DNA strand breakage during physiological apoptosis of the embryonic chick lens: Free 3' OH end single strand breaks do not accumulate even in the presence of a cation‐independent deoxyribonuclease

Chaudun

Arruti²,

Courtois

et al. 1994

Journal Cellular Physiology

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“…The orientation of the inserted DNA was confirmed by restriction fragment mapping (Figs. 1,2). Plasmids with sense (pDOL-25) and antisense (pDOL-28) cDNA orientation, as well as vector without an insert (pDOL-0), were prepared from lysates of transformed Escherichia coli by DNA precipitation followed by cesium chloride density gradient centrifugation.…”

Section: Cell Culturementioning

confidence: 99%

Radiation response of NIH/3T3 mouse fibroblasts overexpressing human poly(adenosine diphosphate ribose) polymerase

Jorgensen

Notario

Thraves

et al. 1993

Radation Oncology Invest

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SUMMARY Poly(adenosine diphosphate ribose) [poly(ADP-ribose)] polymerase is a mammalian enzyme which synthesizes long chains of ADP-ribose attached to various nuclear proteins in response to DNA strand breaks. A role for this enzyme in cellular radioresistance has been postulated due to the radiosensitizing effect of chemical inhibitors of the enzyme on some cell lines. Inhibitor studies, however, lack specificity and direct evidence for involvement of the enzyme in radioresistance is still needed. In experiments described here, intracellular levels of the enzyme were modulated using vectors to express sense and antisense human cDNA transcripts of poly(ADP-ribose) polymerase in stably transfected NIH/3T3 mouse fibroblasts. Although antisense constructs failed to lower enzyme levels, sense transcripts increased enzyme levels. None of the transfectants, however, showed any difference in either radiation sensitivity or DNA strand break repair rates. These results suggest that simple elevation of poly(ADP-ribose) polymerase does not affect the intrinsic cellular radioresistance of these cells. Rudiut Oncol Invest I993;l :I 89-197. 0 1993 Wiley-Liss, Inc.

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“…Regarding the inhibitor kinetics of 3-methoxybenzamide (Figure 6), the 90000-Mr pADPRT fragment from D. discoideum appears to have lost the ability to be stimulated by low doses of inhibitor. This may be due to loss of interaction between multiple binding sites for NADI, which were suggested to cause activation of pADPRT at low concentrations of inhibitors (Jones et al, 1988).…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of NAD+:ADP-ribosyltransferase (polymerizing) from Dictyostelium discoideum

Kofler

Wallraff

Herzog

et al. 1993

Biochemical Journal

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A novel affinity-purification scheme based on the tight binding of NAD+:ADP-ribosyltransferase (polymerizing) [pADPRT; poly(ADP-ribose) polymerase; EC 2.4.2.30] to single-strand nicks in DNA, single-stranded patches and DNA ends has been developed to facilitate the purification of this enzyme from the lower eukaryote Dictyostelium discoideum. Two homogeneous forms of the enzyme, with M(r) values of 116,000 and 90,000, were prepared from D. discoideum by using poly(A) hybridized to oligo(dT)-cellulose as affinity material. The Km is 20 microM NAD+ for the 90,000-M(r) protein and 77 microM NAD+ for the 116,000-M(r) protein. The optimum conditions for the enzyme activity in vitro are 6-10 degrees C and pH 8. The time course is linear during the first 10 min of the reaction only. As in enzymes of higher eukaryotes, the activity is dependent on DNA and histone H1 and is inhibited by 3-methoxybenzamide, nicotinamide, theophylline, caffeine and thymidine.

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Benzamides can stimulate as well as inhibit the activity of nuclear ADP-ribosyltransferase

Cited by 11 publications

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DNA strand breakage during physiological apoptosis of the embryonic chick lens: Free 3' OH end single strand breaks do not accumulate even in the presence of a cation‐independent deoxyribonuclease

DNA strand breakage during physiological apoptosis of the embryonic chick lens: Free 3' OH end single strand breaks do not accumulate even in the presence of a cation‐independent deoxyribonuclease

Radiation response of NIH/3T3 mouse fibroblasts overexpressing human poly(adenosine diphosphate ribose) polymerase

Purification and characterization of NAD+:ADP-ribosyltransferase (polymerizing) from Dictyostelium discoideum

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